The goal of this grant proposal is to further our understanding of the structure and pathophysiologic function of the human TSH receptor (TSHR). We will address the following specific issues:-1. Cleavage of the TSHR: Recent evidence suggests that the large TSHR ectodomain contains two cleavage sites not one as previously thought. We plan to:- (l) Localize the two cleavage sites; (ii) Purify and characterize the putative TSHR C peptide released by intramolecular cleavage; (iii) Determine whether TSHR cleavage occurs in the Golgi complex or after trafficking to the plasma membrane; (iv) Test the hypothesis that TSHR cleavage plays a role in receptor turnover. 2. Overexpression of the TSHR in mammalian cells: Recently, we succeeded in overexpressing the wild-type TSHR in Chinese hamster ovary cells. To provide material for further studies on TSHR cleavage, to stud autoantibody and human TSH interaction with the TSHR, as well as to crystallize a portion of the TSHR ectodomain we wish to attain high level expression of several TSHR variants, including:- (i) a TSHR with a c-myc epitope within the C peptide; (ii) a TSHR cleavage knockout; (iii) Flag epitope-tagged holoreceptors; (iv) Signal transduction deficient holoreceptors; (v) Chimeric TSH-LH/CG receptors selected for their value in discriminating among different types C autoantibodies; (vii) Secreted, soluble TSHR ectodomain variants with 6H epitope tags. 3. Autoantibody and human TSH interaction with the TSHR: We will:- (l) establish direct TSHR autoantibody binding assays using different forms of the TSHR; (ii) complete our mutagenesis studies to localize the region(s) C difference between the human and bovine TSH binding sites on the human TSHR; (iii) explore the role of specie (both ligand and receptor) in assays for TSHR autoantibodies. 4. Role of the 3' untranslated region (UTR) In TSHR expression: The human TSHR 3'UTR plays a major role in suppressing the level of TSHR expression in CHO cells. We propose to examine if the 3'UTR plays a similar role in thyrocytes, to localize the region related to mRNA destabilization, and to investigate putative trans-acting factors that interact with the 3'UTR segment associated with TSHR mRNA destabilization. A similar mechanism in thyroid cells might explain, in part. the very small number of TSHR expressed in thyroid tissue; Also, potential mutations this re ion, b influencing the level of TSHR expression could influence thyroid function.